Multifunctional organic polymers

ABSTRACT

Described herein is a multi-functional polymeric material for use in inhibiting adhesion and immune recognition between cells and cells, cells and tissues, and tissues and tissues. One component of the polymeric material adsorbs well to cells or tissue, and the other component of the polymeric material does not adsorb well to tissues. A water-soluble polymer that does not bear charge (polynonion) is used as the non-binding component, and a water soluble polymer that is positively charged at physiological pH (polycation) is used as the tissue binding component. When the bi-functional polymeric material contacts a tissue, the tissue-binding component binds and thus immobilizes the attached non-binding component, which will then extend generally away from the tissue surface and sterically block the attachment of other tissues. The method and compositions are useful in inhibiting formation of post-surgical adhesions, protecting damaged blood vessels from thrombosis and restenosis, and decreasing the extent of metastasis of attachment-dependent tumor cells.

This is a divisional of prior application Ser. No. 08/132,507 filed onOct. 5, 1993, by Jeffrey A. Hubbell, Donald Elbert, Jennifer L.Hill-West, Paul D. Drumheller, Sanghamitra Chowdhury, and AmarpreetSawhney entitled "Multifunctional Organic Polymers," and now U.S. Pat.No. 5,462,990 which is a continuation-in-part of U.S. Ser. No.07/740,703 filed on Aug. 5, 1991, now U.S. Pat. No. 5,380,536, which isa divisional of U.S. Ser. No. 07/598,880 filed on Oct. 15, 1990, nowU.S. Pat. No. 5,232,984.

BACKGROUND OF THE INVENTION

This invention is generally in the area of organic polymer chemistry,specifically multifunctional polymers.

Cell adhesion plays an important role in human disease. Theseinteractions proceed by the interaction of receptors upon the surface ofa cell with proteins or glycosaminoglycans upon the surface of anothercell or within the extracellular matrix. These receptors may be proteinsor glycosaminoglycans.

Routes to the interruption of these interactions typically involvecompetitive inhibition of these receptor-ligand interactions, forexample, with antibodies (e.g., anti-glycoprotein IIb/IIIa complex foranti-platelet therapy), soluble ligands which act as receptorantagonists (e.g., cyclic RGD peptides or von Willebrand factorfragments), soluble receptors, or other competitors.

It has also recently been demonstrated that it is possible to inhibitthese interactions by mechanical means, for example, byphotopolymerizing poly(ethylene glycol)-based hydrogels upon the cell,cell aggregate, matrix or tissue.

An example of the use of hydrogels to inhibit tissue adhesion isdescribed by U.S. Pat. No. 5,126,141 to Henry. The process utilizesthermo-reversible gels of mixtures of polyoxyalkylene polymers and ionicpolysaccharides applied to the tissues as liquids.

Unfortunately, the inhibitor based methods have a disadvantage common tomany drug therapies, in that it is difficult to restrict the activity ofthe inhibitors to the region of interest. Hydrogel barriers aredifficult to place and it is difficult to control chemical processesassociated with them.

Isolated cells or tissues have also been protected from cell-cellcontact, in this case from attack by immune cells, by placement withinmicrocapsules formed of water soluble non-ionic polymers such aspolyethylene oxide grafted to polycationic polymers such aspoly-L-lysine. However, this is restricted to the use of isolated cellsor tissues which are encapsulated within the polymer at the time ofpolymerization for subsequent implantation into the body.

It is therefore an object of the present invention to provide methodsfor making and using compositions, and the resulting compositions, forinhibiting tissue adhesion and cell-cell contact within the body.

It is a further object of the present invention to provide methods formaking multifunctional polymeric materials which can be biodegradableand which can be used for drug delivery, either at a specifictissue-polymeric material interface or as a result of release ofbioactive agents during degradation of polymeric material.

SUMMARY OF THE INVENTION

A bi-functional polymeric material for use in inhibiting cell-cellcontact and tissue adhesion is disclosed wherein one domain of thematerial (i.e., one region with a particular function) is a polymerwhich adsorbs to cells or tissue (referred to collectively below as"tissue"), and the other domain of the polymeric material is a polymerwhich does not adsorb to tissue. Since most tissues bear a net negativecharge, a positively charged polymer (polycation) is used as thetissue-binding domain. A water-soluble polymer that does not bear charge(polynonion) is used as the non-binding domain. When the two-domainpolymeric material contacts a tissue, the tissue-binding domain(s) bindsand immobilizes the attached non-binding domain(s), which then generallyextends away from the tissue surface and sterically blocks theattachment of other tissues.

Additional domains, linking groups, and bioactive materials can be addedto this basic two-domain structure to confer, for example, adhesion toparticular types of cells or molecules or degradation by enzymatic ornon-enzymatic means. These domains may be a third type of polymer, orwhen serving to direct attachment, a peptide such as RGD, or even asingle amino acid, which is used to target a polyamino acid for cleavageby an enzyme.

The polymer is applied in a fluid phase to the tissues or cells to beprotected, whereupon the tissue binding domains adsorb the polymericmaterial to the tissue. The fluid phase can be applied to isolatedtissue or to tissue during surgery or by means of a catheter or otherless invasive device.

The compositions are useful for blocking adhesion and immune recognitionand thus may be useful in the treatment of many diseases, including theprevention of postoperative adhesions, protecting injured blood vesselsfrom thrombosis and intimal thickening relating to restenosis, anddecreasing the extent of metastasis of tumor cells in tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the interaction between a twodomain polymeric material and cells or tissue which has been treatedwith the polymeric material to decrease adhesiveness.

FIGS. 2a and 2b are photographs of a rat carotid artery 24 hours aftercrush injury without treatment (FIG. 2a) and treated with a 5% solutionof PEG-b-PLL for two minutes (FIG. 2b).

DETAILED DESCRIPTION OF THE INVENTION I. General Structures of thePolymers.

There are three general structures of the polymeric materials describedherein. Each structure is a block copolymer, i.e., a polymer composed ofconnecting multiple polymer chains of different composition. The threestructures are (1) brush copolymers (as in a bottle brush, with abackbone of one composition and bristles of another) with a backbone ofpoly(B) and bristles composed of poly(A), (A)x-b-(B)y; (2) AB blockcopolymers, i.e., (A)x(B)y, or a poly(A) connected at one end to a poly(B); and (3) ABA block copolymers, i.e., (A)x(B)y(A)z, or a poly(A)connected at both ends to poly(A) chains, or in a less preferredembodiment, (B)x(A)y(B)z; where A is a monomer, the polymer of whichdoes not bind strongly to a tissue; B is a monomer, the polymer of whichdoes bind strongly to a tissue; x is an integer of greater than or equalto 5; y is an integer of greater than or equal to 3; and z is an integergreater than or equal to zero. As used herein, "polymeric materials"include polymers of oligomers. X is determined as that number providingthe desired degree of repulsiveness or non-adhesiveness to tissue; y isdetermined as that number providing the desired degree of adhesivenessof the polymeric material to tissues, as discussed in more detail below.Poly(A) and poly(B) are generally linear polymers, although both may belinear or branched. Both A and B can be monomers, macromers or polymers.

A. Selection of "(B) n"

"(B)n" can be any biocompatible water-soluble polycationic polymer, forexample, any polymer having protonated heterocycles attached as pendantgroups. As used herein, "water soluble" means that the entire polymer,poly(B)y, must be soluble in aqueous solutions, such as buffered salineor buffered saline with small amounts of added organic solvents ascosolvents, at a temperature between 20° and 37° C. In some embodiments,poly(B) will not be sufficiently soluble in aqueous solutions per se butcan be brought into solution by grafting with water-soluble poly(A)chains. Examples include polyamines having amine groups on either thepolymer backbone or the polymer sidechains, such as poly-L-lysine andother positively charged polyamino acids of natural or synthetic aminoacids or mixtures of amino acids, including poly(D-lysine),poly(ornithine), poly(arginine), and poly(histidine), and nonpeptidepolyamines such as poly(aminostyrene), poly(aminoacrylate), poly(N-methyl aminoacrylate), poly (N-ethylaminoacrylate), poly(N,N-dimethylaminoacrylate), poly(N,N-diethylaminoacrylate), poly(aminomethacrylate),poly(N-methyl amino-methacrylate), poly(N-ethyl aminomethacrylate),poly(N,N-dimethyl aminomethacrylate), poly(N,N-diethylaminomethacrylate), poly(ethyleneimine), polymers of quaternary amines,such as poly(N,N,N-trimethylaminoacrylate chloride),poly(methyacrylamidopropyltrimethyl ammonium chloride), and natural orsynthetic polysaccharides such as chitosan.

B. Selection of "(A)n"

Any biocompatible, preferably polynonionic at a pH of between 6.5 and8.5, polymer that does not bind to tissue can be used as (A)x. Theseinclude poly(oxyalkylene oxides) such as poly(ethylene oxide),poly(ethyloxazoline), poly(N-vinyl pyrrolidone), poly(vinyl alcohol),and neutral poly(amino acids) such as poly(serine), poly(threonine), andpoly(glutamine) and copolymers of the monomers thereof, polysaccharidessuch as dextran, water soluble cellulose derivatives such as hydroxyethyl cellulose, poly(hydroxyethyl acrylate), poly(hydroxyethylmethacrylate) and polyacrylamide.

In some cases, polymers that bear a net negative charge (polyanion) willalso function well as the non-binding domain, but only in cases wherethe interaction between the polycation and the polyanion is such thatthe two-domain polymer does not precipitate due to the interactionsbetween the opposite charges. When polyanionic polymers are mixed withpolycationic polymers, the mixture often precipitates due to theinteraction of the opposite charges on the two polymers. The extent towhich this occurs depends upon the nature of the charges of theparticular polymers in question, as well as their solvent environment.Such precipitation may also occur with the block copolymers, ifpolyanions are used as the (A)x domains. There may, however, bepolyanion (A)x and polycation (B)y combinations that will notprecipitate, which may be determined experimentally by those of ordinaryskill in the art. In particular, (A)x comprising weak acids such aspolyalcohols may be expected to behave in this way. Polyalcohols aresuch weak acids that they may be considered for the purposes describedherein as being essentially polynonionic. Negatively charged polymersfrom polyacids may also be determined to form stable, soluble copolymerswith suitable polycations in useful aqueous formulations.

It should also be understood that the (A)x and the (B)y blocks couldthemselves be copolymeric, so long as they retain their dominantpolynonionic (for (A)x) or polycationic (for (B)y) character. Forexample, an (A)x could consist of a copolymer of ethylene oxide andanother water-soluble, nonionic comonomer, and a (B)y could consist of acopolymer of lysine and another amino acid with a cationic (e.g.,arginine) or nonionic (e.g., glycine) side chain.

An example of a preferred polymeric material as described herein isformed of poly(L-lysine) (abbreviated PLL), "(B)y", and poly(ethyleneoxide), also known as poly(ethylene glycol) or poly(oxyethylene)(abbreviated PEG), "(A)x". The end groups on the PEG should not beconsidered as specified by the name PEG.

Block copolymers of PLL and PEG are useful two-domain polymericmaterials, where the PEG domain, which does not bind well to tissues, isimmobilized upon a tissue by the adsorption of the PLL, which does bindwell to the tissue.

In general, the molecular weight of (B)y must be sufficient to yield thedesired degree of binding, generally including at least five charges,and having a molecular weight of at least 300 g/mole. In general, themolecular weight of (A)x must be sufficient to provide the desireddegree of repellency of tissue or macromolecules, taking intoconsideration potential steric hindrance of the polymer, generallyrequiring a molecular weight of at least 300 g/mole. The lengths of the(A)x and the (B)y in the domains which would result in good blockage ofadhesive and immune interactions may be determined by routineexperimentation. In general, the larger the ratio of (A)x mass to (B)ymass, the weaker the binding of the polymer and the stronger therepulsion of potentially attaching tissues would be. Thus, the functionof the polymer is used to determine an optimum amount of (B)x requiredto yield good binding and the optimum amount of (A)y to yield goodrepulsion. It should be understood that "good" is a word that must bedefined by the requirements of the particular circumstance at hand,e.g., how long binding is required and how complete a repulsion isrequired by the particular medical application. In addition to the massratio of (A)x to (B)y, the number of chains of (A)x and the number ofchains of (B)y may be varied from many A:B to 1 A:B in (A)x-b-(B)y; to 2A:B in (A)x(B)y(A)z; to 1 A:B in (A)x(B)y), as can the absolute lengthsof the two chains (e.g., an (A)x-b-(B)y with a particular mass fractionof A:B with many short (B)y may function in a different manner than onewith a few long (B)y chains).

II. Specialized Structures of the Polymers.

Modifications of the bi-functional polymers are shown diagrammaticallybelow. The first functionality is the characteristic of tissueadsorbancy, determined by (B)y. The second functionality is thecharacteristic of not adsorbing to tissue, or tissue repellency,determined by (A)x. ##STR1## wherein: A denotes a domain that does notadsorb well to tissues; and

B denotes a domain that adsorbs well to tissues, such as the polycationpoly-L-lysine (PLL).

When present on the tissues, the two-domain polymers will adsorb to thetissues in a conformation that could be graphically represented asfollows, where the solid line represents the tissue surface: ##STR2##

As shown above, the B component adsorbs well to the tissue, and the Acomponent, which has no strong interaction with the tissue, danglesgenerally away from it. The net effect of the adsorption of the dualfunction polymeric material is to link to tissue the A component, whichby itself does not bind well to tissues, thus blocking the adhesion ofother cells, tissues, or macromolecules to the treated surface.

Although described herein with reference to (A)x consisting of a singlepolymer, the structures shown diagrammatically above can be synthesizedusing mixtures of polymers as (A)x. For example, the "bristles" on thebrush polymers could be formed of different (A)x, (A)x₁, (A)x₂, (A)x₃,etc., as could the termini on the AGA block copolymers. Theseembodiments consisting of one or more types of polymers (A)x arereferred to jointly herein as (A)x.

ABA block copolymers are expected to be more effective in repellingtissues than BAB block copolymers, i.e., with a central nonbinding blockand peripheral binding blocks. However, there may be cases where BABblock copolymers demonstrate high binding to the tissues and highdegrees of repulsion of approaching tissues. This is especially truewith the use of strongly binding, short (B)y regions with a long central(A)x region.

A. Control of Duration of Binding.

It may be desirable to control the duration of the repulsiveness of thepolymeric material or the duration of the presence of the polymericmaterial. There are several ways in which this can be accomplished.These are generally divided into (1) selection of (B)y on the basis ofthe strength, or lack of strength, of binding over time to tissue; (2)modification of the polymeric material to incorporate a domain Cresulting in degradation of the polymeric material and/or separation ofthe binding component (B)y from the repulsive component (A)x; andsynthesis of polymeric material where either the binding component (B)yis converted into a non-binding component (D)y or the repulsivecomponent (A)x is converted into a non-repulsive or binding component(F)y.

1. Selection of type and relative ratios of binding and repulsivecomponents.

In the first case, it may be desirable to control the duration of thebinding of the polymer to the treated tissue by controlling its rate ofdesorption. Such control may be desirable to control the duration ofrepulsiveness or to control its presence for regulatory or toxicologicalconcerns. Control can be achieved in several ways. Various (B)y would beexpected to bind to the tissue with different strength. For example, apoly(L-histidine) would be expected to bind to tissues less stronglythan a poly(L-arginine) of the same chain length (i.e., same y in (B)y),because a lower fraction of the histidine residues are charged atphysiologic pH than of the arginine residues. The stronger the bindingof the (B)y, the longer the duration of binding would be. The strengthof binding of various (A)x-b-(B)y, (A)x(B)y, and (A)x(B)y(A)z willdepend upon the particular structure of the polymer and upon the ratioof mass of (A)x to (B)y. In either of these cases, the duration ofbinding is determined by the rate of desorption without any chemicalchanges in the polymer.

2. Incorporation of degradable regions or cleavage sites into thepolymers.

A component which is subject to degradation can be selected to controlthe duration of the presence of or the repulsion of the polymer. Thiscan be accomplished by selecting an (B)y that is degraded bynonenzymatic hydrolysis, such as a polyester, polyamide, polyurethane,or polyanhydride; or that is degraded by enzymatic hydrolysis, such as apolypeptide or polysaccharide. Oxidative mechanisms may also be utilizedto achieve degradation. For example, ether, urethan, and teritary carbonstructures are known to be sensitive to oxidative degradation. The useof these structures in the copolymer may lead to degradation byreactions, e.g., nitric oxide, peroxynitrite, or hypochlorite.Homopolymeric backbones or regions (B)y are expected to havedifferential stability to nonenzymatic or enzymatic degradation in vivo,e.g., poly-L-lysine, poly-D-lysine, polyornithine, polyhistadine, andpolyarginine should display differential stability. For example,poly(D-lysine) would be expected to be more resistant proteolyticdegradation than poly(L-lysine).

The polymeric material can be targeted for cleavage by an enzyme byincluding a recognition amino acid or recognition sequence for an enzymewithin the polymeric material. This targeting component is referred toherein as "C". In general, the enzyme will be a naturally occurringprotease such as trypsin, thrombin, factor XII, factor XIII, othercoagulation factors, collagenase, elastase, plasmin, or otherendoprotease or ectoprotease which recognize specific amino acidsequences. Individual amino acids can be incorporated into the polymerto make the polymer sensitive to degradation by less specific proteases.Saccharide or oligosaccharide regions which are cleaved by a variety ofenzymes present in tissues, such as amylases, could also be used.

Component C could also be selected to be sensitive to nonenzymatichydrolysis, for example, a dimer of lactic acid, the ester of which issensitive to hydrolysis. In this way, the non-binding (A)x is cleavedfrom the (B)y, separating the polymeric material into its twocomponents, one of which no longer binds and the other which no longerrepels tissue. Other nonenzymatically sensitive sites could also beselected, such as other esters, amides, anhydrides, and numerous othersapparent to those skilled in the art of degradable polymers.

Considerable tailoring of this loss of repulsiveness may be obtained bycontrolling how the repulsive bristles fall off via the cleavage ordegradation of component C. This degradation may be controlled by theaction of water without any enzymatic activity, and thus would be solelya function of time, or by the action of an enzyme. Two types of enzymesensitivities could be selected: a sequence that is sensitive to aconstitutively expressed enzyme, e.g., coagulation factor Xa, so thatdegradation would still be mostly a function of time, or one couldselect a sequence that is sensitive to a regulated enzyme, such asplasmin or collagenase, both of which are expressed at the leading edgeof many types of migrating cells during healing. This would allow acell, during healing, to wipe away the repulsiveness that was protectingthe surface prior to healing.

In terms of timing the duration of the presence of the material,degradation of the adsorbing region B would result in removal of thepolymeric material from the surface of the tissue. This may be importantfrom the perspectives of both optimal efficacy and regulatory approval,where the minimum residence of the polymeric material in the body isdesired.

Different embodiments of these modifications can be designed, forexample, the (A)x can be connected to the (B)y via a linker C, to form(A)xC-b-(B)y, (A)xC(B)y, or (A)xC(B)yC(A)z. These embodiments are shownschematically below. ##STR3## wherein C denotes a cleavage site, forexample, for cleavage via enzymatic or nonenzymatic hydrolysis.

3. Selection of (A)x or (B)y which is converted into domains havingdifferent binding affinities.

The duration of binding may also be controlled by designing chemicalsensitivities into the polymers. For example, the backbone of thepolymer may be selected to undergo a nondegradative chemicaltransformation to alter its affinity for the tissue.

In one embodiment, one can obtain desorption by converting the (B)yregion to a nonadsorbing region, (D)y, either by the action of water orthrough the use of a constitutive or regulated enzyme. For example,poly(L-glutamic acid) could be selected as a backbone. This polymer doesnot bind well to tissues since it is polyanionic. It can be converted toa tissue-binding polymer by esterification with a compound such asbromoethylamine hydrochloride, or by amidification with a compound suchas ethylene diamine. The esters and amides are hydrolytically sensitiveand will, with time, be converted back to poly(L-glutamic acid) andethanolamine or ethylene diamine, with the poly(L glutamic acid) regionreadily desorbing. For example, the side chain carboxyls are esterifiedwith ethanolamine (amine protected), so that subsequent hydrolysis ofthe ester in vivo releases the (binding, B) amine to produce a(non-binding, D) carboxyl. Alternatively, the side chain carboxyls maybe amidated with ethylenedianime (one amine protected), and subsequenthydrolysis of the amide in vivo releases the (binding, B) amine toproduce a (nonbinding, D) carboxyl.

This embodiment is shown diagrammatically below: ##STR4## wherein Ddenotes a nonbinding group which arises by reaction of B, a bindinggroup. B converts into D after binding.

In another embodiment, one can design nonadhesive components (A)x thatreact in vivo to yield domains consisting of (E)x that are no longerrepulsive. For example, a polyamine could be selected as the adhesivecomponent. The amine groups could be protected by reaction with acarboxyl-containing group, such as a (hydroxyl protected) hydroxy acid(with subsequent deprotection). The resulting amide bond would renderthe original amine much less adhesive and more, repulsive, yielding theA)x component. Over time, hydrolysis of the amide bond would result inloss of the hydroxy acid, forming the original amine, which wouldincrease adhesiveness and decline in repulsiveness. Such chemicalconversions in vivo could occur nonenzymatically, as described above, oranzymatically, either by a constitutively present or regulated enzyme,as described further above.

This is described diagrammatically below. ##STR5## wherein E denotes anadhesive group, which arose by reaction of A, a repulsive group.

In summary, the overall adsorption of the polymer to a tissue, and thenits repulsion of other tissues, is an optimization: with much (B)y,adsorption will be strong, but subsequent repulsion will be weak; andwith much (A)x, repulsion will be strong, but adsorption will be weak.

There are at least two general ways to determine the duration ofrepulsiveness: (1) by timing the duration of the presence of thepolymeric material on the tissue surface, e.g., by timing degradation ofthe backbone, reaction of (A)x or (B)y to result in desorption, ordesorption without degradation by varying binding affinity of (B)y; and(2) by timing the duration of the repulsiveness of the polymericmaterial on the surface, e.g., by timing the degradation of therepulsive components (A)x or reaction of (A)x to result in loss ofrepulsion.

B. Attachment of Bioactive species.

Additional activities may be added to the polymeric material byattaching bioactive species ("F") to the ends of the (A)x, elsewhere onthe (A)x, or onto the (B)y. Such bioactive species may be adhesionpeptides, adhesion oligosaccharides, antibodies, enzymes, receptorligands, or drugs. Bioactive species may be used to target adhesion ofthe polymeric material, to effect a biological activity at the polymericmaterial-tissue interface, or to effect an activity when released duringdegradation of the polymeric material.

For example, the adhesion peptide Tyr-Ile-Gly-Ser-Arg, from laminin,binds to receptors on endothelial cells, but not on blood platelets.Thus, the addition of the oligopeptide Tyr-Ile-Gly-Ser-Arg to thetermini of the (A)x and adsorbing the polymeric material to a damagedvessel wall would be expected to block thrombosis on the vessel wall butnot to block reendothelialization from the surrounding undamaged vesselwall. This embodiment makes it possible to cover the thrombogenicity ofan injured vessel wall but, via an adhesion ligand F on the termini ofthe (A)x components, to permit the regrowth of endothelial cells uponthe polymer. In this case, F is the pentapeptide Tyr-Ile-Gly-Ser-Arg(YIGSR), which supports endothelial, smooth muscle cell, and fibroblastadhesion, but not platelet adhesion; or the tetrapeptide Arg-Glu-Asp-Val(REDV), which has been shown to support endothelial cell adhesion butnot that of smooth muscle cells, fibroblasts, or platelets, J. A.Hubbell, et al., "Endothelial cell-selective materials for tissueengineering in the vascular graft via a new receptor BioTechnology9:568-572 (1991), the teachings of which are incorporated herein. Thisapproach also permits the reendothelialization of the vessel wall whileit is still not adhesive to platelets, thus enabling healing whileavoiding platelet activation and thrombus formation.

In another embodiment, an enzyme F is coupled to the (A)x termini,thereby covering up the platelet reactivity of the damaged vessel (inangioplasty injury) or mesothelium (in pelvic adhesions) while at thesame time immobilizing a beneficial biological activity. Examples ofuseful enzymes include tissue plasminogen activator (tPA), sinceplasminogen activation is desirable in both prevention ofpelvic/abdominal adhesions and in the resorption of vascular thrombus.Numerous other bioactive species may be beneficial.

This embodiment is shown schematically as: ##STR6## wherein F=Bioactiveagent.

III. Synthesis of Polymers

In general, methods for synthesis of the different polymeric materialswill be apparent to those skilled in the art from the foregoingdescription.

A. Brush Block copolymers: 1.Method for grafting A upon the ε amines ofB, with all the ε amines deprotected, using stoichiometric control inwater.

PEG may be bonded to the ε-amines of lysine residues of poly(L-lysine)as follows. Poly(L-lysine) (PLL) is reacted with monomethoxy PEG, theterminal hydroxyl of which has been previously activated withcarbonyldiimidizole (CDI). The PLL and the activated PEG are mixed in anaqueous solution buffered at pH 9 and allowed to react for 48 hours atroom temperature. The number of PEG chains grafted per PLL chain may becontrolled by the ratio of moles activated PEG added per mole PLL added.The reaction may not proceed to completion, i.e., the mole ratio of PEGto PLL in the reaction mixture may not be identical to that in thePEG-b-PLL product, but higher ratios of PEG to PLL will produce higheramounts of PEG in the PEG-b-PLL product.

2. Method for grafting A onto the ε amines of B, with all the ε aminesdeprotected, using stoichiometric control in anhydrous solvent.

The cationic domains tend to be highly reactive, and efforts must bemade to control the extent of addition of "A" to "B". Executing thereaction in the absence of water reduces deactivation of "A" and allowsbetter stoichiometric control. For example, unprotected poly-L-lysine isdissolved in water, then added to dimethylformamide (DMF) to make asolution that is 5% aqueous. The poly-L-lysine is then reacted with CDImono-activated PEG in stoichiometric amounts, followed by evaporation ofsolvent under vacuum yielding an (A)x-b-(B)y copolymer. Alternatively,unprotected poly-L-lysine is dissolved in water and is precipitated byaddition of NaOH. The precipitated polymer is added to anhydrous DMF andthen reacted with CDI mono-activated PEG in stoichiometric amounts,yielding an (A)x-b-(B)y copolymer. When the reaction is performed in theabsence of water, side reactions involving the activated group arereduced (i.e., deactivation is reduced), and at long reaction times theratio of mole PLL to PEG in the polymer product more closely resemblesthan in the reactant mixture.

3. Grafting A upon the ε amines of B, with a controlled number of thoseamines deprotected.

Solution polymerization of PLL may be carried out using monomerscontaining different epsilon protecting groups, which allows strictcontrol over the degree of substitution of "A" onto "B". N-carboxyanhydrides of various amino acids may be synthesized and polymerizedinto copolymers, as in the following example.N,N'-dicarbobenzoxy-L-lysine (Z,Z-lysine) is reacted with phosphoruspentachloride to yield ε,N-carbobenzoxy-α,N-carboxy-L-lysine anhydride.α,N-carbobenzoxy-ε,N-tert-butyloxycarbonyl-L-lysine (Z,boc-lysine) isreacted with sodium methoxide to yield the sodium salt of Z,boc-lysine.The sodium salt of Z,boc-lysine is reacted with phosphorus pentachlorideto yield ε,N-tert-butyloxycarbonyl-α,N-carboxy-L-lysine anhydride.Z,Z-lysine anhydride is added to Z,boc-lysine anhydride, and the twomonomers are polymerized by the addition of sodium methoxide as aninitiator. A copolymer results, poly(ε boc-lysine)-co-(ε Z-lysine). Theboc groups are removed by addition of the polymer to trifluoroaceticacid for fifteen minutes. The salt form is converted to the free base byreaction with a reactant such as pyridine. The free amines on thepolymer are then reacted with CDI PEG in DMF. The Z groups aredeprotected by addition of the polymer to HBr in acetic acid for fifteenminutes, yielding an (A)x-b-(B)y copolymer, where the ratio of PEG toPLL in the final product is controlled by the inital ratio of bocprotected lysines, which were ultimately grafted with PEG, to Zprotected lysines, which were not grafted.

As an alternative to solution polymerization, greater control over thelength of the "B" segment may be obtained through the use of solid phaseorthogonal synthesis. For example, poly-L-lysine is synthesized using apeptide synthesizer. The monomer lysines used in the synthesis are oftwo types. One type of lysine is α-amine protected using the F-mocprotecting group (9-fluorenylmethyloxycarbonyl group), and is ε-amineprotected with the Z protecting group (benzyloxycarbonyl group). Thesecond type of lysine is α-amine protected using the F-moc protectinggroup, and is ε-amine protected with the t-Boc protecting group (tert.butyloxycarbonyl group). A thirty residue peptide is constructed, withone t-boc protected lysine per nine Z protected lysines. The t-bocgroups are selectively cleaved by immersion of the peptide in neattrifluoroacetic acid for fifteen minutes, and then desalted withpyridine. The free amines are reacted with CDI mono-activated PEG inDMF. The Z groups are then deprotected with HBr in acetic acid for 15minutes, yielding an (A)x-b-(B)y copolymer.

4. Solid phase synthesis of PLL copolymerized with another residue, theside groups of which are used for grafting of A.

Not only may the protecting groups be varied, but the type of residuemay also be changed. For example, on a peptide synthesizer, a peptide ismade utilizing both Z protected lysine and tert. butyl protectedaspartic acid in a ratio of nine lysines per one aspartic acid. Thetert. butyl group is deprotected by immersion of the polymer in neattrifluoroacetic acid. The free carboxyl groups are activated withO-(N-Succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSU)in DMF. The TSU activated peptide is reacted with mono-amine-PEG in DMF.The Z protecting groups on the lysines are removed by incubation of thepeptide with HBr in acetic acid, yielding an (A) x-b- (B) y copolymer.

B. AB (and ABA) Block Copolymers: 1. Grafting A to the terminal amine(and carboxyl) prior to deprotection of the ε amines.

It may be desirable to produce versions of the polymer which are not ofa brush structure. This may be facilitated by not deprotecting theepsilon amines of PLL, so that the only reactive groups are the amineand carboxyl termini. For example, reaction of CDI mon-activated PEGwith poly ε,N-carbobenzoxy-L-lysine in DMF yields an (A)x-(B)ycopolymer. Activation of the carboxyl terminus of the (A) x-(B)ycopolymer with TSU followed by reaction with mono-amino PEG in DMFyields an (A)x-(B)y-(A)z copolymer.

2. Solid phase synthesis of PLL using two monomer lysines with differentamine protection chemistries, the N (and C) terminal residue(s) beingdifferently protected than the other lysine residues.

The presence of a free amine on both ends of the "B" segment wouldrequire fewer reactions to attach "A" segments to the ends of thepolymer. This may be done by beginning the synthesis with a residuecontaining an epsilon protecting group which may be removed withoutdisturbing the other epsilon protecting groups. The "A" chain reactswith this free amine, as well as the free amine at the N terminus of thepeptide. For example, poly ε,N-carbobenzoxy-L-lysine is synthesized bysolid phase methods, and the ε-residue at the carboxyl terminus is bocprotected, with all other ε-residues Z protected. The boc is removed intrifluoroacetic acid, and the free amines are reacted with CDImono-activated PEG in DMF. The Z groups are removed with HBr in aceticacid, yielding an (A)x-(B)y-(A)z copolymer.

3. Solid phase synthesis of PLL copolymerized with another residue, theN (and C) terminal residues being the different composition, the sidegroup(s) of which will be used for grafting of A.

Beginning the synthesis of a pure poly-L-arginine chain with a lysinewill place a reactive amine at both ends of the poly-L-arginine chain.The poly-L-arginine would be the "B" binding segment, and "A" segmentscould be placed onto the amines. For example, poly-L-arginine issynthesized by solid phase methods, but the residue at the carboxylterminus is a boc protected lysine. The boc group is removed intrifluoroacetic acid, and the peptide is reacted with CDI PEG in pH 9buffered water, yielding an (A)x-(B)y(A) z copolymer, with the (B)ysegment representing poly-L-arginine.

The following examples are examplary of methods of synthesis for thespecialized modified embodiments of the polymeric materials.

4. Solution phase polymerization of N-carboxyl anhydride lysine with PEGas initiator to yield AB copolymer.

In the solution phase polymerization of a polyamino acid, the sodiumsalt of the PEG may be used as an initiator. For example, thepolymerization of the N-carboxy anhydride of lysine is initiated by thesodium salt of monomethoxy PEG, as described by Pratten, M. K., Lloyd,J. B., Horpel, G., Ringsdorf, H., "Micelle-forming block copolymers:Pinocytosis by macrophages and interaction with model membranes",Makromol. Chem. 186, 725-33 (1985).

5. Polymerization of ethylene oxide with PLL as initiator.

Ethylene oxide is polymerized to PEG by the addition of an initiator,with incorporation of the initiator into the polymer. For example:Ethylene oxide polymerization is initiated by the addition of polyε,N-carbobenzoxy-L-lysine, which leads to PEG-co-polyε,N-carbobenzoxy-L-lysine.

C. Incorporation of a degradative or cleavage component, C, into thepolymer.

The time of residence of the polymer on the tissue and the ability ofthe host to clear the polymer may be enhanced by incorporating sequenceswhich are non-enzymatically degradable.

1. For degradation at bristles by addition of a C component.

C can be added to the polymeric material so that the "bristles" arereleased from the backbone when the C component degrades. For example,monomethoxy PEG reacts with d,l-lactide (1:3 molar ratio) in xylene inthe presence of stannous octate under reflux for sixteen hours to yielda PEG with an end group which degrades over time in water. The hydroxylat the terminus of the trilactide end group is activated with CDI, whichis then further reacted with PLL by methods presented above to yield an(A)xC-b-(B)y, an (A)xC-(B)y or an (A)xC-(B)y-C(A)z copolymer.

2. Incorporation of C into the backbone for enzymatic degradation of thebackbone.

Certain amino acid sequences are recognized by specific proteolyticenzymes. These may be incorporated into a polypeptide backbone. Forexample, by solid phase methods, a peptide is produced which containssix lysines, followed by the sequence proline-X-glycine-proline, where Xis any neutral amino acid, and repeated three times to obtain a peptidewith 30 residues. The sequence proline-X-glycine-proline is hydrolysedby the enzyme collagenase. The peptide is reacted with CDImono-activated PEG to yield an (A)x-b-C(B)y, an (A)x-C(B)y or an(A)x-C(B)yC-(A)z copolymer which is enzymatically degradable.

D. Synthesis of a polymeric material using a component that changes fromone function to a second function over time. 1. Synthesis of a polymericmaterial using a B component that converts from a binding component to anon-binding component D.

The duration of binding of a polymeric material can be controlled bysynthesis of a polymeric material with a non-binding backbone which isconverted to a binding backbone through degradable linkages. Forexample, terminal amine polyglutamic acid is reacted with CDI PEG toproduce an (A)x(D)y-b-(B)y copolymer. The copolymer is dissolved inwater at pH 2 and lyophilized to convert the carboxylic acid salt to thefree acid. The polymer is dissolved in DMF, and the glutamic acidresidues are activated with TSU. The activated polymer is then reactedwith boc protected aminoethanol in DMF overnight at room temperature andthen deprotected and desalted. The product is initially polycationic andbinding, but hydrolyses to a non-binding polyanion.

2. Synthesis of a polymeric material using an A component that convertsfrom repelling tissue to one which does not repel or bind to tissue.

A polypeptide may be reacted with an (unprotected) hydroxy acid usingpeptide synthesis techniques to yield a nonionic polymer. Over time,this amide linkage degrades, and the nonion converts from repulsive tonot repulsive. For example, using solid phase synthesis, a peptide isproduced in which the first twenty residues are lysines, the next tenresidues are arginine, and the next twenty residues are lysines. Thepeptide is fully deprotected, and is then reacted with lactic acid,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), andN-hydroxysulfosuccinimide in buffered water at pH 9, yielding an(A)x-(B)y-(A)z copolymer in which the "A" segment is originallynonionic, but upon degradation becomes ionic.

E. Addition of bioactive components to the polymeric material.

Some interactions of tissues with the polymer may be desirable. Avariety of mechanisms can be used to effect this result, including cellspecific peptide sequences that can be used to target the polymer tobind only to certain cell types, or to attact or inhibit binding ofsoluble mediators, including enzymes.

1. Incorporation of cell specific peptide sequences.

Cell specific peptide sequences can be incorporated into the copolymeras follows. The peptide tyrosine-isoleucine-glycine-serine-arginine(YIGSR) is fully protected with the exception of the carboxyl terminus,and is activated with2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU). This is then reacted with mono-amino PEG in DMF, givingα-hydroxy, ω-YIGSR PEG. This compound is CDI activated in DMF, reactedwith PLL in DMF, followed by deprotection of YIGSR in DMF, yielding acopolymer with a pendant bioactive agent.

2. Derivatization of PEG with an amino acid.

The derivitization of PEG with an amino acid makes it suitable forinclusion into a polypeptide using solid phase techniques. For example,CDI mono-activated PEG is reacted with α-Fmoc protected lysine, formingε-lysine-PEG. Using solid phase techniques, a copolymer is made oflysines and ε-lysine-PEGs, using the method of Atassi, M. Z., Manshouri,T., "Synthesis of Tolerogenic Monomethoxypolyethylene Glycol andPolyvinyl Alcohol Conjugates of Peptides", J. Prot. Chem., 10, 623-7(1991), the teachings of which are incorporated herein.

III. Medical Applications of Polymers

As described above, the bi-functional polymers have a variety ofapplications. Several are demonstrated in detail in the followingexamples. These include local application, either at the time of surgeryor via injection into tissue, to prevent adhesion of tissues; to deliverbioactive compounds where release is effected more efficiently or at amore desirable rate or where tissue encapsulation could detrimentallyeffect or delay release; prevention of thrombus formation at bloodvessel surfaces, particularly following angioplasty; alteration ofattachment of cells, especially to prevent attachment of cells, andtherefore decrease metastasis of tumor cells; and as coatings onprosthetic implants such as heart valves and vascular grafts derviedfrom processed tissues.

As defined herein, "tissue" includes cells, cell aggregates, tissuesremoved from the body, and tissues present in the body. This term canalso be applied to treated tissue, such as tissue heart valves, bloodvessels and membranes, where the tissue is no longer living and has beenchemically fixed, or a cryopreserved blood vessel or other tissue.

The polymeric materials can be applied directly by localized or topicalapplication, or if targeted as described herein, systemically. Topicalor localized application is achieved generally by spraying or injectinga very thin layer (usually on the order of monolayers of polymericmaterial) onto the tissue surface to which adhesion is desired. Methodsfor applying the polymeric materials in this manner are known to thoseskilled in the art.

As described below in the examples, these polymeric materials have beenapplied in three systems where the prevention of cell and tissue contactis desirable.

1. Damage to the surface of organs during surgery may cause adherence oftissues which are in close contact. This problem is particularly severein reproductive surgery, where extensive adhesion formation may causeinfertility. In a surgical model in rats, an injury was made to theuterine horns, followed by treatment with PEG-b-PLL. Adhesions werereduced from an extent of 78% for control (treated with HEPES-bufferedsaline, the vehicle) to 9% for treatment with a 1% concentration of thepolymeric material by peritoneal lavage.

2. After balloon angioplasty, damage to the endothelium may lead tothrombus formation, due to the interaction of platelets with the vesselwall. Moreover, thrombosis may lead to restenosis. In rats, an injurywas made to the carotid artery, followed by treatment with PEG-b-PLL.Thrombosis was completely blocked by exposure to a 5% concentration ofthe polymeric material prior to resumption of blood contact.

3. During laparoscopic removal of tumors, tumor cells may be releasedinto the pelvic cavity, and the tumor cells may metastasize. Tumor cellswere incubated with PEG-b-PLL, and then seeded intraperitoneally inmice. Implantation of tumor cells upon the injured tissue surfaces wasreduced from 0.93 g in controls to 0.17 g in treated animals.

There are a number of other medical applications for the polymericmaterials described herein.

1. Thrombosis upon injured vascular surfaces may cause complications invascular surgery. This is especially true in microvascular surgery,where thrombosis at the ends of the vessels at the anastomosis may leadto occlusion. Prior to anastomosis, the ends of the vessels to be joinedare dipped in a 1% solution of PLL-b-PEG, or a similar bi-functionalpolymeric material, to pacify the injured vessel surfaces. Thrombosismay thus be reduced, and vessel patency may be improved.

2. Cardiovascular implants are adversely affected by thrombosis,platelet attachment and aggregation. As an example, thrombosis upon thesurfaces of heart valve implants made from human or animal tissuesprocessed, for example, by cryopreservation, lyophilization or chemicalfixation, is implicated in acting as a nidus for calcification. Thus,thrombosis may lead to long-term valve failure by calcification of thevalve to materials. Tissue heart valves are placed in a 2% solution ofPEG-b-PLL, or a similar bifunctional polymeric material, in bufferedsaline at 4° C. for 1 hour. The valve is then rinsed in saline for 5minutes and packaged for implantation later. During the incubation inthe polymeric material solution, the bi-functional polymeric materialadsorbs by its binding domain, thus immobilizing the non-binding domainin the processed tissue valve. This subsequently blocks thrombosis afterimplantation, and long-term calcification at the sites of thrombosis maythus be reduced.

3. The adhesion of immune cells to the vascular surface of atransplanted organ is one of the causes of acute organ rejection. Thisacute rejection may be related to an acute inflammatory reaction, inwhich white blood cells adhere to the vascular surface of thetransplanted organ. Hearts for transplantation are placed in apreservation medium containing added bi-domain polymeric material, suchas PEG-b-PLL. The organ is stored in the medium, e.g., at 4° C., untiltransplantation. During the storage period, the bifunctional polymericmaterial adsorbs to the vascular surface of the organ, whichsubsequently blocks the adhesion of immune cells to the organ aftertransplantation. This may reduce acute rejection of the organ.

4. In many cases, it may be advantageous to have a drug that isimmobilized upon and or released from a surface. An example is thefibrinolytic enzyme streptokinase (SK). Streptokinase is consideredinferior to tissue plasminogen activator (tPA) because tPA binds tofibrin and exerts its fibrinolytic activity in an immobilized manner. SKexhibits no such binding. This difficulty can be eliminated by bindingof the enzyme to the polymeric material described herein. SK is attachedto the ends of the non-binding domains which are connected to a bindingdomain. In this case, since the bioactive group is rather large, beingan enzyme, a fewer number of nonbinding domains is optimal, as is alarger binding domain. The SK-grafted bi-domain polymeric material,e.g., SK-grafted PEG-PLL AB block copolymer, is administered locally tothe site of balloon angioplasty using a catheter, such as a ballooncatheter that weeps fluid. The SK-PEG-PLL binds to the vessel surface,yielding a localized and more sustained fibrinolytic activity exactly atthe site of its application.

The same technology can be applied using any of numerous other classesof drugs, such as growth factors, growth factor antagonists, receptorantagonists, adhesion factors, adhesion antagonists, antimitotics,antisense oligonucleotides, and many others. In a similar approach, thedrug is attached to the ends of the nonbinding domain via a clearablelinker, such as a lactic acid dimer or oligomer, or an enzymaticallycleavable peptide or saccharide. In this way, a controlled release ofthe tissue-bound drug may be engineered into the conjugate of the drugwith the bi-functional polymer.

5. In vascular injury, such as occurs when a diseased coronary artery isinjured by balloon angioplasty, the endothelial monolayer that protectsthe vessel wall from contact with blood is removed. Thrombosis followsdue to blodd platelet binding to proteins in the injured vessel wall.This interaction can be blocked using bi-functional polymeric materials,such as poly(N-vinyl pyrrolidinone)-chitosan block copolymers,abbreviated PVP-chitosan. The ratio of the PVP to chitosan, and themolecular configuration of the PVP-chitosan, which is dependent on thenumber of PVP per chitosan, may be optimized to resist substantially alladhesion of blood cells to the vessel wall. This blocks thrombosis,which is suspected to cause further vessel disease such as restenosis.It may also slow the process of reendothelialization. A bioadhesivedomain may be attached to the ends of the nonbinding domains, and thisbioadhesive domain may be selected to bind to endothelial cells andsupport their adhesion but not to blood platelets so as to supportthrombosis. The laminin pentapeptide tyr-ile-gly-ser-arg (YIGSR) isattached via its N-terminal primary amine to end-groups on thePVP-chitosan AB block copolymer. After adsorption, the nonbinding groupsblock thrombosis, but the YIGSR peptide on the termini of the nonbindinggroups support the attachment and migration of endothelial cells fromthe noninjured periphery of the blood vessel. This may thus acceleratevascular healing and permit reendothelialization of the injured zonewithout contact of the artery with blood. Such approaches may be carriedout on numerous other tissues, e.g., a demesothelialized surface toaccelerate peritoneal healing and prevent adhesions, and adeepithelialized cornea to accelerate healing after corneal abrasion,among others.

6. In some cases the binding domain may be highly specific in nature,rather than nonspecific as with exclusively polycationic bindingdomains. For example, a water-soluble polymer that is nonionic, such aspoly(vinyl alcohol) (PVA), may be grafted to a receptor antagonist, suchas a receptor antagonist that binds to adhesion receptors on cellsurfaces. For example, a PEG-PVA AB block copolymer is synthesized.Subsequently, several sialyl Lewis-X (LeX) oligosaccharides are attachedto the multiple alcohols on the PVA chain. This PEG-LeX-PVA copolymer isadministered by injection into the blood stream. The polymeric materialbinds to the surfaces of cells expressing E-selectin, which binds toLeX. Since there are several LeX oligosaccharides bound to the PVAdomain, binding to E-selectin on cell surfaces is quite strong. Thus,the LeX PVA domain serves as the binding domain, and the PEG domainserves as the nonbinding domain. The net effect may thus be to block theadhesion of cells via numerous receptors by immobilizing a nonbindingdomain such as PEG to the surface of the cell.

IV. Optimization of the Polymeric Material for Individual Applications.

The biological performance of these materials is optimized by alteringthe structure of the polymers, the ratio of the number of tissue-bindingpolymers to non-binding polymers, and the ratio of the mass of thetissue-binding polymers to non-binding polymers.

In some case, polymeric materials exhibiting more than one manner ofdegradation may be required to achieve different results. For example,degradation by nonenzymatic hydrolysis will depend primarily upon theaccessibility of the polymeric material to water and the local pH. Giventhat pH and water concentration are similar throughout many parts of thebody, such a mode of degradation would yield a loss in repulsivenessthat depends mostly upon time. As another example, if the degradableregion was sensitive to an enzyme, the activity of which was not highlyregulated but rather was present in the body fluids at a more or lessconstant level, the rate of loss of repulsiveness would again dependprimarily upon time. As another example, if the degradable region wassensitive to an enzyme, the activity of which was more highly regulated,the rate of loss of repulsiveness would then depend more upon theexpression of that particular enzyme activity. For example, many typesof cells express the proteases plasmin or collagenase during migration.A sensitivity to plasmin would then cause all migrating cells to degradethe repulsiveness of the polymeric material and thus attach. This mightbe useful, e.g., in protecting an injured vessel wall from bloodplatelets, but permitting an endothelial cell migrating from anoninjured region of the vessel wall to migrate over the treated area,degrading its nonadhesiveness as it did so and permitting theendothelial cell to attach to the vessel wall and migrate over it. Thiscould permit reendothelialization of a de-endothelialized vessel wallwithout ever permitting direct contact of the endothelial cell-freesurface with blood platelets.

The biological performance of these polymeric materials depends upontheir structure. Specific features of biological performance includebinding to the tissue, repulsion of opposing tissues, duration ofbinding to the tissue, duration of repulsion of opposing tissues, andthe mode of loss of binding or repulsion. Specific features of polymericmaterial structure include the type (chemical composition) oftissue-binding domain, type of non-binding domain, the ratio of the massof binding to non-binding domains, the number of binding to non-bindingdomains, the inclusion of sites that are particularly susceptible tononenzymatic hydrolysis, the inclusion of sites that are particularlysusceptible to enzymatic hydrolysis, and the inclusions of sites withparticular biological affinity.

A variety of ways can be utilized to optimize the desired properties.For example, when a PEG-b-PLL brush copolymer is used to protect aninjured tissue surface from the adhesion of cells approaching from thefluid phase in contact with that tissue surface, the polymeric materialcan be made more desirable by studies conducted using a tissue culturemodel, such as cell adhesion to gelatin coated surfaces in tissueculture medium containing 20% serum. Gelatin is coated upon themultiwell dishes using standard tissue culture technique, for example,from a 3% gelatin solution. A 30 minute exposure to medium with 20%serum will lead to the adsorption of fibronectin, a protein leading tocell adhesion., to the gelatin surface. If fibroblasts are seeded onthis surface in medium containing 20% serum, rapid adhesion andspreading will occur. A measurement of the fraction of cells adhering(Fa) and fraction of cells spreading (Fs) may be made based onmorphological criteria using light microscopy. Such measurementsconducted 4 hours following seeding provide useful measures of adhesionand repulsion. A family of PEG-b-PLL polymeric materials aresynthesized, e.g., according to the following schedule:

    ______________________________________                                                              Fraction of lysine residues                             MW of PLL  MW of PEG  grafted with a PEG chain                                ______________________________________                                         1000      1000       1/50                                                     1000      1000       1/10                                                     1000      1000       1/5                                                     10000      1000       1/50                                                    10000      1000       1/10                                                    10000      1000       1/5                                                     10000      5000       1/50                                                    10000      5000       1/10                                                    10000      5000       1/5                                                     10000      10000      1/50                                                    10000      10000      1/10                                                    10000      10000      1/5                                                     20000      5000       1/50                                                    10000      5000       1/10                                                    20000      5000       1/5                                                     20000      10000      1/50                                                    20000      10000      1/10                                                    20000      10000      1/5                                                     ______________________________________                                    

For each polymeric material composition, Fa and Fb are measured at 4hours. Polymeric materials with very low ratio of PEG to PLL, such asthe polymeric material with 20000 Da PLL, 5000 Da PEG, with 1/50 of thelysine residues grafted with a PEG chain, would be expected to berelatively ineffective in prevention of fibroblast attachment andspreading.

In the case where a brush copolymer which desorbs slowly from tissue isdesirable, the same assay system could be used, and polymeric materialscould be synthesized with other binding domains, such as in the schedulebelow:

    ______________________________________                                                                         Fraction of                                              MW of                binding residues                             Binding domain                                                                            binding              grafted with a                               composition domain   MW of PEG   PEG chain                                    ______________________________________                                        Poly L lysine                                                                             20000Da  5000Da      1/10                                         Poly L histidine                                                                          20000    5000        1/10                                         Poly L arginine                                                                           20000    5000        1/10                                         Poly L ornithine                                                                          20000    5000        1/10                                         Poly        20000    5000        1/10                                         ethyleneimine                                                                 Poly amino  20000    5000        1/10                                         ethylacrylate                                                                 Chitoson    20000    5000        1/10                                         ______________________________________                                    

By varying the specific nature of the charge, in this example used toobtain binding to tissues, the rate of desorption may be adjusted. Inthe assay system, cells could be seeded once each day on a gelatinsurface variously treated, and Fa and Fs measured the 4th hour afterseeding. Afterwards, the substrates could be rinsed with medium andstored in tissue culture with medium for examination after the next day.Subsequent measurements over many weeks would permit the rate of loss ofcell repulsiveness to be determined for each formulation.

In the situation where an AB block copolymer was to be used to preventthrombosis and intimal thickening upon an artery surface injured byballoon angioplasty, after initial experimentation as described above todetermine a desirable ratio of binding domain to non-binding domain, anda desirable optimal composition of the binding and non-binding domains,animal experimentation could be performed. For example, if the aboveexperimentation yielded good repulsion for 14 days with PLL of degree ofpolymerization (DP) 30 and a PEG of DP 150, animal experimentation couldbe performed making small excursions about this formulation, for exampleby the following schedule:

    ______________________________________                                               PLL DP PEG DP                                                          ______________________________________                                               20     100                                                                    30     100                                                                    40     100                                                                    20     150                                                                    30     150                                                                    40     150                                                                    20     200                                                                    30     200                                                                    40     200                                                             ______________________________________                                    

Thrombosis could be measured at 24 hours following injury and treatment,and intimal thickening could be measured at 28 days following injury andtreatment, using light microscopy with histological staining withGomori's trichrome staining for the measurement of thrombosis andVerhoeff's staining for the measurement of intimal thickening.

If it were desired to further optimize, for example based on the natureof the binding domain, an optimal formulation from the above schedulecould be selected, and the PLL domain could be replaced withpoly(L-histidine), poly(L-arginine), poly(L-ornithine),polyethyleneimine, poly(aminoethylacrylate), and chitosan, with guidancefrom the experimentation in vitro.

If it were desired to further optimize based on the rate of degradationof the binding domain, an optimal formulation from the above schedulecould be selected and the poly(L-amino acid) binding domain could bereplaced with a poly(D-amino acid) domain of the same amino acid, forexample poly(D-lysine) for poly(L-lysine). Each polyamino acid would beexpected to exhibit different stability to proteolysis in vivo.

If it were desired to even further optimize based on the rate ofdegradation of the binding domain, an optimal formulation from the aboveschedule could be selected and a protease sensitive site could be placedbetween the binding and the non-binding domains. For example, many cellsexpress proteases on their leading edge as they migrate, such asplasmin, collagenase, or elastase. By appropriate selection of aprotease sensitive site it should be possible to design a polymericmaterial that is sensitive to endothelial migration, permitting thepolymeric material to prevent thrombosis, but also permitting thepolymeric material to be degraded by endothelial cells repopulating thevessel surface as healing and reendothelialization occurs.

A similar outcome may be obtained by placing adhesion peptides at thetermini of the non-binding domains. For example, the laminin peptidetyr-ile-gly-ser-arg is known to support endothelial cell adhesion andspreading. However, platelets do not bind avidly to this sequence. It isthus possible to synthesize an AB block copolymer with the adhesionpeptide on the termini, or elsewhere, on the non-binding domain. Thismay also permit the prevention of platelet contact while at the sametime permitting endothelial cells to migrate over the injured vesselzone to repopulate and reendothelialize the injured zone.

An AB block copolymer can be used to immobilize a drug upon a tissuesurface. For example, the formation of fibrin is known to be involved inthe formation of postoperative adhesions, and fibrinolytic proteins havebeen shown to be effective in reducing adhesion formation. Moreexpensive fibrinolytic proteins such as tissue plasminogen activator(tPA) have advantages over less expensive ones such as streptokinase(SK) in that the tPA binds to the tissue surface at sites of fibrin.This retention of tPA by immobilization is an important benefit. Thisretention may be mimicked, however, by attaching the SK to a bi-domainpolymeric material. For example, a family of AB block copolymer could besynthesized according to the following schedule:

    ______________________________________                                        DP of PLL    DP of PEG  Presence of SK                                        ______________________________________                                        25           100        Yes                                                   50           100        Yes                                                   100          100        Yes                                                   25           500        Yes                                                   50           500        Yes                                                   100          500        Yes                                                   ______________________________________                                    

These polymeric materials could be evaluated for the ability to preventpostoperative adhesion in rats after devascularization injury of theuterine horn, each delivered as a 1% solution in buffered saline. Afterselection of an optimal formation, the equivalent polymeric materialcould be synthesized with albumin, an inactive protein, rather than SKto serve as a control.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1: Synthesis and Characterization of PEG-b-PLL. Materials andMethods. PEG-b-PLL Synthesis.

Poly(ethylene glycol) was grafted to poly(L-lysine) based on thecalculations of Sawhney and Hubbell, "Pol;y(ethyleneoxide)-graft-poly(L-lysine) copolymers to enhance the biocompatibilityof poly(L-lysine)-alginate microcapsule membranes" Biomaterials13:863-870 (1992), and on the chemistry of Beauchamp, et al., "A newprocedure for the synthesis of polyethylene glcol-protein aducts: theeffects on function, receptor recognition, and clearance of superoxidedismutase, lactoferrin, and alpha 2 macroglobulin" Anal. Biochem.131:25-33 (1983), the teachings of which are incorporated herein.Monomethoxy terminated poly(ethylene glycol((PEG) (20 g, mol. wt. 5000;Aldrich, Milwaukee, Wis., USA) was dried by azeotropic distillation frombenzene, and precipitated by addition to anhydrous diethyl ether. Thepolymer was dried overnight under vacuum, and stored under argon. Theterminal hydroxyl on the PEG was then activated with1,1-carbonyldiimidazole (CDI; Sigma, St. Louis, Mo.). Under argon, CDI(0.194 g) was weighed out, then added to dry PEG (10 g) in anhydrousdichloromethane. The reactants were stirred under argon at roomtemperature for two hours, and the product was then precipitated byaddition to anhydrous ether. The product was dried under vacuumovernight.

Poly(L-lysine) (PLL) (1 g, mol. wt. 21,400; Sigma) was dissolved in 25ml of 50 mM sodium borate buffer (pH 9) and filter sterilized threetimes through 0.2 μm syringe filters (Nalgene, Rochester, N.Y.) toremove microbial contamination. In a sterile reaction bulb and in asterile cabinet, CDI activated PEG (CDI-PEG) (6.6 g) was added to thepoly(L-lysine) solution with vigorous stirring for one day. The solutionwas then dialyzed (SpectraPor™ dialysis tubing, MW cutoff 12,000-14,000;Spectrum Medical Industries, Los Angeles, Calif.) against four liters ofphosphate buffered saline (pH 7.4) for one day. The solution was thenfreeze dried, yielding 3.44 g of product.

NMR Analysis of PEG-b-PLL.

¹ NMR analysis of PEG-b-PLL was performed in D₂ O on a 500 MHz GeneralElectric instrument.

Results

¹ NMR analysis of PEG-b-PLL in D₂ O indicated the presence of new peakswhich were not present in the individual spectra of PLL or PEO. Thesepeaks corresponded to the conversion of some epsilon amines on the PLL.The new peaks were presumably due to the addition of PEG to the PLL.Analysis of the peak at 2.9 ppm, corresponding to the protons on thedelta carbon on the PLL side chain, and a smaller peak at 3.0 ppm,corresponding to the protons on delta carbons of side chains which hadreacted with CDI-PEG, indicated that 5.73% of the lysyl residues hadreacted with CDI-PEG. This corresponds to 9.57 PEG chains per PLL chain,or an average molecular weight of PEG-b-PLL of 69,250 Daltons.Comparison of the peaks at 2.9 ppm and 3.0 ppm with the peak at 3.3 ppm,resulting from the monomethoxy group on the PEG, indicated that only19.3% of the PEG in the product was actually attached to the PLL. Thiswas due to incomplete dialysis of the product, however, the presence ofthe PEG could be accounted for in the experiments by the choice ofappropriate controls.

EXAMPLE 2: Effect of PEG-b-PLL on cell spreading monomethoxy PEG off onone capped end in vitro. Materials and Methods.

Human foreskin fibroblasts (HFFs) were incubated in DMEM containing 10%fetal bovine serum (FBS) with 0.2% PEG-b-PLL (as described in Example1), PLL or PEG for five minutes, and were then seeded into 24 welldishes which also contained DMEM with 10% FBS and 0.2% (w/v) of theappropriate polymer. The fibroblasts were seeded at 2000, 10,000, 25,000or 40,000 cells/cm², with two replicates at each concentration. At threehours post seeding, the number of well spread cells and the number oftotal cells was counted at 200× on a Nikon phase contrast invertedmicroscope, two fields per well, and the percentage of cells which werewell spread was calculated.

Results.

The percentage of spread cells (spread cells/total cells×100) is anindication of seeding efficiency. Seeding efficiency will remainrelatively constant regardless of concentration of cells within therange of cell concentrations studied here. However, a polymer whichprevents spreading at low concentrations may be ineffective at higherconcentrations. Thus, it is logical to examine a range of cell seedingconcentrations.

                  TABLE 1                                                         ______________________________________                                        Prevention of Fibroblast Spreading                                                   concentration of cells (cells/cm.sup.2                                        2000    10,000    25,000    40,000                                     polymer  % spread cells; std. dev.                                            ______________________________________                                        control  50.8 ± 18.4                                                                          37.5 ± .71                                                                           39.5 ± 9.19                                                                        59.0 ± 5.66                           MPEG     33.8 ± 26.9                                                                          56.0 ± 1.41                                                                          59.5 ± 12.0                                                                        62.5 ± .71                            PLL      5.75 ± 7.22                                                                          23.5 ± 7.78                                                                          27.5 ± 3.54                                                                        41.0 ± 1.41                           PEG-b-PLL                                                                               0.0 ± 0.0                                                                            1.0 ± 1.41                                                                           0.5 ± .71                                                                          1.5 ± .71                            ______________________________________                                    

PLL seems to inhibit spreading somewhat of HFF's at low cellconcentrations, however, at higher seeding concentrations, PLL allowedcell spreading. PEG-b-PLL strongly inhibited fibroblast spreading at allconcentrations tested.

EXAMPLE 3: In vivo study of the use of PEG-b-PLL to prevent pelvicadhesions. Materials and Methods.

PEG-b-PLL (as described in Example 1) was tested at a 1% concentrationin 10 mM HEPES buffered saline. From gravimetric analysis, it wasestimated that PEG-b-PLL was 20% PLL by weight. Thus, 0.2% PLL and 0.8%PEG were mixed together in 10 mM HEPES buffered saline as a control,referred to as the PLL+PEG control. To control for the individualeffects of PLL and PEG, a 0.2% solution of PLL, referred to as the PLLcontrol, and a 0.8% solution of PEG, referred to as the PEG control,were prepared, both in 10 mM HEPES buffered saline. HEPES bufferedsaline alone was also tested, referred to as the HEPES control.

The polymer solutions were prepared as follows: The 10 mM HEPES bufferedsaline was first autoclaved and then filter sterilized (150 mldisposable filter system, Corning, Corning, N.Y.) before addition topolymers. Polymer solutions were shaken until dissolved, and thensyringe filter sterilized as above. The solutions containing PLL,however, were filter sterilized four times to assure removal ofmicrobial contamination. The polymer solutions looked identical, andwere labeled by a code to assure that the surgeries would be performedblind.

Controlled injuries to the uterine horn in 14 week old rats were made byelectrocautery to devascularize the horns and produce serosal injury onthe antimesenteric surfaces. Seven days after surgery, the animals weresacrificed and the horns were examined. The portion of the horn whichwas judged to be adhered to mesentery or rectum was measured with aruler, and compared to the total length of the horn. This "percentadhesion" refers to the length of the horn which displayed adhesionscompared to the total length of the horn. In each animal, the "percentadhesion" values were averaged between the two horns. This average valuewas considered to be one data point.

The uterine horns were judged to be free of adhesions (grade 0),containing filmy adhesions (grade 1) or containing severe adhesions(grade 2). If only a portion of an adhesion was found to be grade 2, thelength of the grade 2 area was measured, and compared to the length ofthe adhesion which was grade 1. It was thus possible to score a hornwith an intermediary value between grade 1 and grade 2. The grade ofadhesions in the animals were averaged between the two horns.

Data for percent adhesion of horn was analyzed using a one factoranalysis of variance. Data for severity of adhesions was analyzed usingthe distribution free, non-parametric Kruskal-Wallis test.

Results.

The extent of adhesions as assessed seven days after uterine horn injuryis presented in Table 2.

                  TABLE 2                                                         ______________________________________                                        Extent of Adhesions                                                           Treatment  ave. % adhesion                                                                              Std. Dev.                                                                              Count                                      ______________________________________                                        HEPES      77.9           6.62     7                                          MPEG       34.4           3.77     6                                          PLL        26.0           4.76     7                                          PEG + PLL  20.3           8.10     7                                          PEG-b-PLL  8.79           4.09     7                                          ______________________________________                                    

All treatments differ significantly from other treatments at 95% by theFisher PLSD post-hoc test, except for PLL versus PEG+PLL, which did notdiffer significantly.

The extent of adhesions was reduced by the application of PEG, PLL orPEG-b-PLL as compared to the HEPES alone control. The combination of PEGplus PLL did not differ significantly from PLL alone. The PEG-b-PLLtreatment was significantly different from the PEG+PLL treatment.

The severity of adhesions was also assessed and is presented in Table 3.The data gathered concerning the grade of the adhesions was parametric,but not normally distributed. The average grade was calculated, and isindicative of the severity of the adhesions. The standard deviation isuseful to judge the reproducibility of the data, despite the lack ofstatistical meaning due to the non-normal distribution. The data wasanalyzed using a rank sum type test. The rank sum from each treatmentwas divided by the number of samples in the treatment, yielding the meanrank.

                  TABLE 3                                                         ______________________________________                                        Severity of Adhesions                                                         Treatment  ave. grade  std. dev.                                                                              mean rank                                     ______________________________________                                        HEPES      1.57        .45      26.7                                          PEG        1.08        .20      17.0                                          PLL        1.03        .07      16.1                                          PLL + PEG  1.07        .19      16.6                                          PEG-b-PLL  .821        .31      10.9                                          ______________________________________                                    

The Kruskal-Wallis test suggested a statistical difference betweentreatments, with p=0.0128 H(corrected for ties)=12.71.

EXAMPLE 4: In Vivo Study: Prevention of Thrombosis following ArteryCrush using PEG-b-PLL. Materials and Methods.

Male Sprague-Dawley rats weighing 350-400 g were used in this study. Theanimals were anaesthetized with pentobarbital (50 mg/kg body weight).Xylocaine was administered subcutaneously in the region of the surgicalmanipulations just prior to incision. The carotid artery was exposed bycareful dissection 1 cm. beyond the external carotid branch point.Atraumatic arterial clips were used to clamp off the artery at the mostproximal exposed point of the internal and external carotid branches andat the most distal exposed point of the common carotid. PE50polyethylene tubing attached to a 30 gauge needle was introduced intothe internal carotid. Residual blood was rinsed from the artery usingHEPES buffered saline. The common carotid was crushed with hemostats toinduce an injury to the arterial wall. The artery was then filled with asterile 5% solution of PEG-b-PLL (as described in Example 1) in HEPESbuffered saline. The solution remained in the vessel for two minutes andthen was withdrawn through the tubing. The tubing was removed, and theinternal carotid was tied off with 5-0 silk suture. The arterial clipswere then removed to allow blood flow. The muscular layer of theincision was closed with continuous 3-0 Vicryl suture, and the skin wasclosed with 9 mm staples. Animals were sacrificed at either two hours ortwenty-four hours post-surgery. The vessels were excised, rinsed, andfixed in buffered formalin. At least twelve hours later, the vesselswere dehydrated in a graded ethanol series, exchanged with xylene,embedded in paraffin, sectioned to a thickness of 5 μm, and mounted onglass slides. Staining was by Masson's trichrome.

Results.

As expected in the case of an arterial wall injury, all control animalsdisplayed significant thrombosis formation, both at the two andtwenty-four hour time points. Application of the PEG-b-PLL solutionprevented thrombosis at both time points. All treated arteries werecompletely patent, allowing normal blood flow through the carotid artery(at 2 hour time point, n=2 treatment, n=2 control; at 24 hour timepoint, n=3 treatment, n=2 control; figures shown are typical of all nfor that condition).

FIGS. 2a and 2b are photographs at a magnification of 600 fold of therat carotid artery 24 hours after crush injury without treatment (FIG.2a) and treated with a 5% solution of PEG-b-PLL for two minutes (FIG.2b). There were no signs of adverse tissue response to bi-functionalpolymer.

EXAMPLE 5: Effect of PEG-b-PLL addition on Tumor Cell Seeding in vivo.Materials and Methods.

A model of laproscopic surgery was performed. A 16 gauge angiocatheterwas inserted intraperitoneally in male C3H mice, and insufflated with 5ml of air. Three treatments were performed, with 18 animals pertreatment. Treatment 1 consisted of the injection of 1×10⁶ tumor cellsin one ml of phosphate buffered saline. Treatment 2 consisted ofinjection of 1×10⁶ tumor cells in one ml of phosphate buffered salinecontaining 0.1% PLL-b-PEG. Treatment 3 consisted of the injection of1×10⁶ tumor cells in one ml of phosphate buffered saline containing 1%PLL-b-PEG. After sixteen days, the tumor volume was compared at thetrocar site, as well as at all other sites.

Results.

The results are presented in Table 4. Application of PLL-b-PEG reducedtumor volume significantly (p<0.05). The differences between 0.1% and 1%PLL b PEG were not significant.

                  TABLE 4                                                         ______________________________________                                        Tumor Volume at 16 days                                                                                  All Other                                          Treatment      Trocar Site (g)                                                                           Sites (g)                                          ______________________________________                                        control        .485        .929                                               .1% PLL-b-PEG  .179        .297                                               1% PLL-b-PEG   .160        .173                                               ______________________________________                                    

EXAMPLE 6: Synthesis of methacrylic PEG copolymerized with aminoethylmethacrylate.

Monomethoxy PEG was reacted with methacryloyl chloride under anhydrousconditions, producing methacrylic PEG. This was copolymerized withaminoethyl methacrylate (AEMA), to yield 85 AEMA:15 methacrylic PEG. Theproduct was then dialyzed against water using the method of U.S. Pat.No. 5,075,400 to Andrade, et al. "Polymer supersurfactants for proteinresistance and protein removal".

EXAMPLE 7: Evaluation of AEMA/methacrylic PEG copolymer in vitro.

Human foreskin fibroblasts were trypsinized and seeded in complete mediacontaining 0.2% (w/v) AEMA/methacrylic PEG copolymer. Cells were seededon polystyrene culture dishes at a concentration of 2000, 15,000, 30,000cells/cm².

                  TABLE 5                                                         ______________________________________                                        Concentration of Spread Cells 3 hour                                          post-seeding.                                                                            concentration of cells (cell/cm.sup.2)                             treatment    2000       15,000    30,000                                      ______________________________________                                        AEMA copolym.                                                                                0            0         0                                       none         212        >1000     >1000                                       ______________________________________                                    

The results demonstrate the effectiveness of the polymer in preventingattachment and growth of the treated cells, especially as compared withthe control cells.

Modifications and variations of the methods and compositions describedherein will be obvious to those skilled in the art from the foregoingdetailed description. Such modifications and variations are intended tocome within the scope of the appended claims.

We claim:
 1. A method for making a biocompatible polymeric material themethod comprisingforming a biocompatible copolymer which is selectedfrom the group consisting of copolymers having the formulas (A)x(B)y;(A)x(B)y(A)z; (B)x(A)y(B)z; and a brush copolymer (A)x-(B)y havingbristles of poly(A); wherein (A)x, (A)y and (A)z are biocompatiblesynthetic polymers and mixtures of polymers that form a region which ispolynonionic at a pH of between 6.5 and 8.5 and does not bind tissue;and wherein (B)y, (B)x, and (B)z are biocompatible, water-solublesynthetic polymers or mixture of polymers that form a region which ispolycationic at a pH of between 6.5 and 8.5 and binds to tissue; andwherein x is an integer of greater than or equal to 5, y is an integerof greater than or equal to 3, and z is an integer of greater than orequal to 0; wherein the polymer has a molecular weight of at least 300g/mole.
 2. The method of claim 1comprising reacting the polymersselected from the group consisting of (A)x, (A)y, (A)z, (B)x, (B)y and(B)z under conditions wherein the polymers are chemically coupled toform the copolymer.
 3. The method of claim 2 wherein (A)x and (A)z areselected from the group consisting of poly(oxyalkylene oxides),poly(ethyloxazoline), poly(N-vinyl pyrrolidone), poly(vinyl alcohol),neutral poly(amino acids), and copolymers of monomers selected from thegroup consisting of oxyalkylene oxides, ethyloxazoline, N-vinylpyrrolidone, vinyl alcohol, and amino acids.
 4. The method of claim 2wherein (B)y is selected from the group consisting ofpoly(ethyleneimine), quaternary amines, and polyamines having aminegroups on either the polymer backbone or the polymer sidechains.
 5. Themethod of claim 1 further comprising covalently incorporating into thepolymeric material a region C that is subject to degradation in vivo byhydrolysis, enzymatic degradation, or oxidation.
 6. The method of claim5 wherein C is selected from the group consisting of peptide sequencesand saccharide sequences cleaved by an enzyme.
 7. The method of claim 5wherein C is selected from the group of chemical compounds whichhydrolyze in the presence of water.
 8. The method of claim 5 wherein Cis selected from the group of chemical compounds which oxidize in vivo.9. The method of claim 5 wherein the C region or regions is incorporatedbetween the tissue binding regions (B)y and the non-tissue bindingregions (A)x.
 10. The method of claim 5 wherein the C region is locatedwithin the tissue binding region (B)y between the points of attachmentof the non-tissue binding regions (A)x.
 11. The method of claim 1wherein the tissue binding region (B)y is selected to convert to anon-tissue binding region when exposed to water, oxidation or toenzymatic attack.
 12. The method of claim 11 wherein the tissue bindingregion (B)y is formed by polymerization of amides or esters.
 13. Themethod of claim 1 wherein the non-tissue binding region (A)x is selectedto convert to a tissue binding region when exposed to water, oxidationor to enzymatic attack.
 14. The method of claim 1 further comprisingincorporating into the polymeric material an agent which is biologicallyactive in a patient.
 15. The method of claim 14 wherein the polymericmaterial is biodegradable, and the agent is a component which isreleased as the polymeric material degrades.
 16. The method of claim 14wherein the agent is chemically coupled to the polymeric material. 17.The method of claim 1 further comprising incorporating the polymericmaterial within a pharmaceutically acceptable carrier for administrationto a patient.